Estrogen receptor a (ERa) is the major therapeutic target for breast cancer, and ERa binding at target genes in breast cancer cells is guided by DNA binding pioneer factor proteins such as FoxA1. Although tamoxifen is a successful therapeutic for suppressing ER-mediated gene regulation, resistance to tamoxifen occurs in most breast cancer patients within five years and breast cancer remains one of the leading causes of women's death worldwide, highlighting the critical need for improved strategies. With an estimated annual breast cancer therapeutic market of $3-5 billion, the market is receptive to directed strategies. A major challenge in ERa drug design is the necessity to identify small-molecules that specifically target the molecular mechanisms of aberrant gene regulation in breast cancer, while leaving the physiological benefits of ERa action intact. The most critical interaction for ERa-mediated gene transcription occurs at its estrogen response element (ERE) DNA site, and FoxA1 enhances ERa binding at EREs in breast cancer cells. However, DNA-directed therapeutics focused on modulating the ERa-FoxA1-DNA interaction are under- studied areas of drug design because technologies to comprehensively examine this interaction are not yet amenable to high throughput drug discovery. We have developed Cognate Site Identifier (CSI) DNA microarrays, containing every permutation of a 10 to 12 base pair DNA sequence within duplex DNA, and Sequence Specificity Landscapes (SSLs) as a tool to analyze the million-plus data points resulting from the CSI array. This proposal will focus on understanding the comprehensive DNA binding specificity of FoxA1- ERa in breast cancer and specifically disrupting FoxA1 enhancement of ERa binding with DNA-directed peptide nucleic acids (PNAs). The specific aims of this Phase I proposal are: 1. The DNA binding preferences of FoxA1 will be characterized using FoxA1 from breast cancer cell lysates and as a purified protein, by CSI-SSL analysis. These studies will identify direct DNA binding sites of purified FoxA1 as well as potential protein-tethered FoxA1-DNA interactions in breast cancer cells. 2. DNA-binding PNAs will be designed to specifically target and disrupt the FoxA1-DNA interaction, as model therapeutics to down-regulate FoxA1-mediated transcription at genes instrumental in breast cancer regulation. 3. A high throughput screening platform will be developed using CSI DNA microarrays to examine the ERa- FoxA1-DNA interaction and screen test compounds as targeted disruptors of this interaction. Our findings will define a new arena for DNA-based therapeutics by expanding CSI-SSL technology to a DNA-bound heterodimeric protein complex, towards small-molecule screening, discovery of novel drug targets, and development of targeted DNA binding molecules as therapeutics for breast cancer. PUBLIC HEALTH RELEVANCE: Estrogen receptor alpha (ERa) is the major therapeutic target for breast cancer, and ERa binding at target genes in breast cancer cells is guided by DNA binding pioneer factor proteins such as FoxA1. While tamoxifen represents a first-line pharmaceutical defense for ERa-positive breast cancer, the field is in critical need of directed therapies because approximately 30% of patients are initially recalcitrant to tamoxifen and, of those who do respond, virtually all will become resistant to tamoxifen within five years. We are developing new technologies, the Cognate Site Identifier DNA microarray and Sequence Specificity Landscape, as a high throughput platform to rapidly identify compounds that specifically target and disrupt ERa-FoxA1-DNA interactions, for discovery of novel DNA-directed breast cancer drugs.